Methods for treating chemoresistant cancer-initiating cells

ABSTRACT

The disclosure provides methods of treating cancer by selectively inhibiting p-S 552 -β-catenin, p-T 217 -β-catenin, T 332 -β-catenin, and/or p-S 675 -β-catenin production and/or activity. Such methods also and/or limit cancer-initiating cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application Ser. No. 62/198,670, filed Jul. 29, 2015, thedisclosure of which are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION Field of the Invention

The disclosure provides methods of treating cancer by selectivelyinhibiting p-S⁵⁵²-β-catenin, p-T²¹⁷-β-catenin, p-T³³²-β-catenin, and/orp-S⁶⁷⁵-β-catenin production and/or activity. Such methods also reduceand/or limit cancer-initiating cells.

Description of Related Art

The cancer stem cell (CSC) model proposes that tumors are maintained bya unique sub-population of cells with self-renewal capacity thatdifferentiate into mostly non-tumorigenic progeny. Some evidencesuggests these often rare CSCs preferentially survive standardchemotherapeutic treatments, but distinguishing CSCs from the bulk ofthe tumor and specifically targeting them remains a considerablechallenge. The recent renaissance in understanding the role of clonalevolution in tumorigenesis illuminated the challenge of acquiredresistance and has led to two major models of tumorigenesis—the cancerstem cell and clonal evolution. These are not necessarily mutuallyexclusive and can be complementary. Both indicate that the main obstacleto durable cures is cancer heterogeneity. However, existing anti-cancertherapy largely fails to account for either model. Recent advances intargeted therapy are promising, but since cancer heterogeneity andevolution present multiple, moving targets, development of resistance iscommon. Unfortunately, little progress has been made in targeting thechemoresistant cells responsible for relapse. However, regardless ofwhether clonal evolution, cancer stem cell, or a hybrid model bestexplains therapeutic resistance, it has been noted that stemness mayrepresent a critical target. Stemness refers to the molecular programsthat govern and maintain the stem cell state, and central to this stateis the ability to self-renew. Absent self-renewal, cancers cannotpersist or regenerate following chemotherapy, but understanding and thentargeting self-renewal remains an unmet challenge.

The link between tumorigenesis and aberrant self-renewal is illustratedby the Wnt/β-catenin and PI3K/Akt signaling pathways. Wnt signalingplays a prominent but complicated role in these processes. Severalstudies demonstrate a critical role for β-catenin in CSCs and indicatethat CSCs can be targeted through β-catenin pathway inhibition, butsuggest that this alone is not sufficient for eliminating tumors. ThePI3K/Akt pathway, which is negatively regulated by the tumor-suppressorPTEN, is frequently dysregulated in cancer due to its central role incell proliferation, growth, survival, and metabolism as well as stemcell regulation. Extensive efforts have focused on pharmacologicallyinhibiting this pathway for anti-cancer therapy. Nonetheless, emergingclinical data have shown only limited efficacy for PI3K pathwayinhibitors, and animal studies showed that PI3K inhibitor treatmentcould lead to the outgrowth of resistant clones. Indeed, using a Ptenmutant T-cell acute lymphocytic leukemia (T-ALL) mouse model, it wasshown that rare, self-renewing chemoresistant leukemic stem cells (LSCs)identified as lineage negative (Lin⁻) CD3⁺c-Kit^(Mid) cells and theirbulk blast cell progeny had differential sensitivity to differenttargeted treatments. The Wnt/β-catenin and PI3K/Akt pathways have evenbeen shown to cooperate in tumorigenesis. Pten deletion results inintestinal polyposis caused by excessive intestinal stem cell activity.Mechanistically, this effect is driven in part by Akt phosphorylation ofβ-catenin at serine 552 (pS⁵⁵²-β-catenin), leading to β-cateninactivation. Indeed, β-catenin was shown to confer resistance to PI3K andAkt inhibitors and promote metastasis in colon cancer. The tankyraseinhibitor XAV-939, an indirect inhibitor of β-catenin, was shown toreverse this resistance in vitro. Unfortunately, low activity of thisinhibitor in vivo precludes effective clinical use.

SUMMARY OF INVENTION

Targeting the Wnt/β-catenin and PI3K/Akt pathways for more effectiveanti-cancer activity offers the potential but also the limitations asnoted above. The inventors have found that that the Wnt/β-catenin andPI3K/Akt pathways cooperatively interact to promote HSC self-renewal andexpansion. While activation of either pathway individually was notcompatible with long-term self-renewal—with Pten deletion resulting inHSC proliferation but exhaustion due to excessive differentiation andβ-catenin activation blocking differentiation but resulting in apoptosisof HSCs—in ombination, the two cooperatively drove self-renewal and HSCexpansion by blocking differentiation and apoptosis in proliferatingHSCs (Perry, J. M. et al. Cooperation between both Wnt/β-catenin andPTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cellself-renewal and expansion. Genes Dev 25, 1928-1942 (2011), incorporatedby reference). Pharmacological agents stimulating both pathways ingenetically normal HSCs led to expansion of untransformed stem cells;however, permanent, genetic activation of both pathways resulted inleukemic transformation.

Multiple lines of evidence indicate that self-renewing CSCs/LSCs areresponsible for chemoresistance. Because the Wnt/β-catenin and PI3K/Aktpathways interact to stimulate self-renewal and through phosphorylationof β-catenin by Akt (pS⁵⁵²-β-catenin), targeting pS⁵⁵²-β-catenin mayinhibit oncogenic self-renewal. In the methods of the disclosure, thenormal self-renewal was stimulated to discover an inhibitor of oncogenicself-renewal.

Thus, in broad aspect, the invention provides methods of treatingcancer, comprising administering to a subject in need thereof apharmaceutically active molecule that is capable of selective)inhibitin9 p-S⁵⁵²-β-catenin, p-T²¹⁷-β-catenin, p-T³³-β-catenin, and/orp-S⁶⁷⁵-β-catenin production and/or activity, wherein thepharmaceutically active molecule is administered in an amount effectiveto reduce and/or limit cancer-initiating cells.

Surprisingly, the inventors found that doxorubicin (DXR, or Doxo, orDOX), a long-used chemotherapeutic agent, selectively inhibitspS⁵⁵²-β-catenin with minimal effect on total β-catenin. At high dosestypically used in the clinic, DXR acts as a DNA-damaging agent byinhibiting topoisomerase II. DXR and other chemotherapeuticspreferentially target tumors, and DXR has such broad and efficaciousanti-cancer activity relative to other chemotherapeutics. The inventorsfound that, by using low, metronomic doses of DXR, particularly throughslow-release, long-circulating DXR nanoparticles (NanoDXR or NanoDoxo),leukemia-initiating activity of LSCs can be inhibited while sparingHSPCs. In vivo, this treatment reduced pS⁵⁵²-β-catenin levels in LSCs,prevented LSC expansion, essentially eliminated LSC tumorigenicactivity, and was accompanied by recovery of hematopoieticstem/progenitor cells (HSPCs, Lin⁻Sca1⁺c-Kit⁺) and substantiallyincreased survival. The inventors also found a dynamic relationshipbetween rare LSCs and their bulk leukemic blast cell progeny in responseto cytotoxic chemotherapy. Notably, it was found that binary targetingof bulk leukemic blasts with cytotoxic chemotherapy and chemoresistantLSCs by targeting pS⁵⁵²-β-catenin-dependent oncogenic self-renewal isnecessary for optimal survival. In distinguishing the unique propertiesof LSCs and their progeny, the inventors found that both populationsmust be differentially targeted at both the ‘root’ and ‘branch’ ofcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that cooperative activation of the Wnt/β-catenin andPI3K/Akt pathways successively expands HSPCs, LSCs and T-ALL blastcells. Pten:β-cat^(Act) mice were induced by tamoxifen. a, At 9 wpi,flow cytometry analysis of BM showed that all double mutants, but notsingle mutants, developed leukemia characterized by ≥20% CD45^(Hi) blastcrisis cells. b, These cells predominantly expressed CD3 but lacked bothCD4 and CD8 expression, indicative of an early T-ALL. c, Prior to T-ALLdevelopment, Pten:β-cat^(Act) mice exhibited expansion of HSPCsidentified by flow cytometry as Lin⁻Sca-1⁺c-Kit⁺ (LSK) cells. d, TheHSPC population collapsed as LSCs, identified as Lin⁻c-Kit^(Mid) CD3⁺cells, expanded. e, Kaplan-Meier survival curves indicated that alldouble mutants, but not single mutants, succumbed to leukemia by 12 wpi.f, Anti-pS⁵⁵²-β-catenin antibody staining (dark gray) of control, singleand double mutant spleens counterstained with light H&E at 8 wpi.Frequencies are based on percent of total nucleated cells±standarddeviation (SD). Scale bars, 10 μm.

FIG. 2 shows that DXR inhibits the pSer⁵⁵² active form of β-catenin. a,Flowchart summarizing HTS design. b, Activity of compounds fromvalidation library was tested against HEK-293 TOPFlash Akt^(Act)βcat^(Act) (TOP) and control HEK-293 FOPFlash Akt^(Act) βcat^(Act) (FOP)cells at multiple doses for inhibition of luciferase activity.Cytotoxicity profiles (CTG) of compounds were also determined. Shown arerepresentative data from 3 compounds of interest. Dose-response data (b)were used to calculate the effective concentration of compoundsresulting in 90%, 50%, and 25% inhibition of luminescence orcytotoxicity (EC₉₀, EC₅₀, and EC₂₅) using nonlinear regression analysis.TOP and FOP cells were treated with candidate compounds at EC₉₀, EC₅₀and EC₂₅ derived from (b) for 48 hours, washed and flash frozen forWestern analysis (c). See Methods for additional detail. d,Computational model showing predicted binding of Akt and DXR toβ-catenin. e-f, FRET analysis verifying interaction between AKT andβ-catenin. Cells transfected with EGFP-AKT and mCherry-β-catenin weretreated with vehicle or 25, 50, 100, and 200 nM Doxorubicin, and FRETefficiency was determined (e). f, FRET efficiency at 0.5, 1, 2 and 3 hrspost-doxorubicin (200 nM) addition.

FIG. 3 shows that chemotherapy-induced high expression ofpS⁵⁵²-β-catenin in LSCs is inhibited by low-dose DXR treatment. a,Schematic of experimental setup for leukemia treatment mouse modelsystem. b, Illustration of strategy for repurposing DXR as a targetedtherapy. Open arrows indicate a single treatment cycle for typicalclinical use of DXR and targeted use strategy (inner box) drawn torelative scales. Triangles represent DXR treatment drawn proportionallyto scale. The cumulative targeted dose (distributed over 5 daysconsecutively) is indicated to relative scale by the inner triangle(white) (see Methods). c, Representative flow cytometry sorting gatesindicating HSPC, LSC and (non-LSC) T-ALL blast cell populations. d,Populations from (c) were sorted from leukemic mice treated as indicatedin (a,b) with vehicle, chemotherapy, [Low]DXR or chemotherapy+[Low]DXRand stained with Anti-pS⁵⁵²-β-catenin antibody. Mean fluorescentintensity (MFI) is indicated for each population and treatment. e,Representative images of pS⁵⁵²-β-catenin staining of LSCs sorted frommice treated as indicated from (d). Scale bars, 30 μm (inset 3 μm).

FIG. 4 shows differential response of LSCs and blast cells tochemotherapy and [Low]DXR treatment. Leukemic mice were treated withvehicle, chemotherapy, [Low]DXR or chemotherapy +[Low]DXR as describedin FIG. 3a . a-c, At 10 days post-treatment, BM was analyzed by flowcytometry to determine frequency of blast cells (a), LSCs (b) and HSPCs(c). Shown are representative plots. d, Average frequency±SD of eachpopulation from a-c (n 6 per group). e, Limiting-dilution assays todetermine CRU frequency were performed on blast cells and LSCs sortedfrom chemotherapy treated leukemic mice and on HSPCs sorted from[Low]DXR treated mice. Engraftment 1% blast cells) was determined in NSGrecipients at 10-12 weeks post-transplant.

FIG. 5 illustrates chemotherapy induction combined with maintenancepS⁵⁵²-β-catenin inhibition markedly improves therapeutic outcome. a, b,Cohorts of leukemic mice were treated with vehicle, chemotherapy,[Low]DXR or chemotherapy+[Low]DXR as in FIG. 3a . At 12 dayspost-treatment, BM was harvested from treated mice and transplanted intosub-lethally irradiated NSG recipients. a, Treatment schematic andKaplan-Meier curves of recipient mice. b, Recipients of BM from [Low]DXRonly treated leukemic mice were analyzed by flow cytometry at 6 monthspost-transplant. Shown are representative plots of blast cells, LSCs,and HSPCs with average frequency±SD of surviving 29/30 recipients fromthis group. c, Treatment schematic and Kaplan-Meier curves of micetreated as indicated after leukemia development. d, LSC and HSPCfrequency in BM of leukemic mice at 10 days post-treatment withchemotherapy and either free [Low]DXR or [Low]nanoDXR. e, Treatmentschematic and Kaplan-Meier curves of chemotherapy +weekly [Low]nanoDXRtreatment for 10 weeks total. Dashed line indicates day of final[Low]nanoDXR treatment. f, Surviving [Low]nanoDXR treated mice wereanalyzed by flow cytometry at 230 days post-treatment.

FIG. 6 illustrates hematopoietic lineage analysis in control, Pten,β-catAct, and Pten:β-catAct mice. a, Percentage of immature (B220Low,IgM+), mature (B220High, IgM+) and Pre-Pro B (B220Low, IgM-) cells incontrol, single and double mutant bone marrow 8-9 wpi as determined byFACS analysis. b, Percentage of Mac-1+ Gr1+ myeloid cells in bone marrow(top) and spleen (lower) in 8-9 wpi control, single and double mutantsas determined by FACS analysis. c, FACS diagrams illustrating controland double mutant bone marrow analysis of T-cell lineage cellsquantified in d. d, Percentage of CD3+, double and single positive Tcells in control, single and double mutant bone marrow at 8-9 wpi. Notethe logarithmic scale. e-f, Double Negative (DN) populations in control,single and double mutant thymus at 8-9 wpi. Representative FACS plots ofcontrol (upper panel) and double mutant (lower panel) thymus is shown ine. g-h, Double and single positive thymocyte populations from control,single and double mutants. Representative FACS plots of control (leftpanel) and double mutant (right panel) thymus is shown in g. Results aregraphed as mean±SD.

FIG. 7 illustrates Bone Marrow and spleen histology of Pten:β-catActleukemic mice. a, Femur of control (left) and Pten:β-catAct mutants(right). H&E sections of bone marrow diaphysis and trabecular boneregion of epiphysis. b, Representative spleens from control, single anddouble mutants. 1 cm scale bar. c, Masson's Trichrome stained sectionsof spleens from b.

FIG. 8 illustrates inhibition of β-catenin prevents expansion of LSCs inresponse to chemotherapy but increases morbidity. a-b, Double mutantmice were treated with chemotherapy (Nelarabine+dexamethasone) with orwithout pan-β-catenin inhibitor. Flow cytometric analysis revealed thatchemotherapy stimulated the expansion of LSCs. Additional treatment withpan-β-catenin inhibitor prevented this expansion; however, β-catenin'scritical role in normal cellular function results in poor survival (c).

FIG. 9 shows FRET verification between Akt and β-catenin. While FRET wasobserved in mCherry-β-catenin+EGFP-AKT transfected cells and could beinhibited by DXR, essentially no discernible FRET occurred whenmCherry-β-catenin was transfected with EGFP alone (see also FIG. 2e-f ).

FIG. 10 illustrates that DXR preferentially inhibits LSC expansion invitro. a-b, BM isolated from leukemic Pten:β-catAct mice at 8 wpi wascultured in HSC expansion media. Doxorubicin, 0105375, and thioguanosinewere added to 11, 33 or 100 nM and cultured for 72 hours and analyzed byflow cytometry for LSCs (a) and HSPCs (b) as in FIG. 1. Fold changebefore and after culture for each population is indicated relative toequivalent vehicle control concentrations.

FIG. 11 illustrates normal dose DXR acts similar to chemotherapy inreducing blast cells but [Low]DXR does not. a, Leukemic mice establishedas described in FIG. 3a were treated with vehicle, chemotherapy(Nelarabine (Nel.) +dexamethasone (Dexa.)), [Low]DXR with Dexa., ornormal dose DXR (8x higher than [Low]) with Dexa. At 10 dayspost-treatment, BM was analyzed by flow cytometry to determine frequencyof blast cells. Average frequency±SD (n≥6 per group). Note that unlikeFIG. 4, this experiment used normal or [Low] DXR as a substitute forNelarabine but Dexamethasone treatment was retained in all groups due tothe inability of single DNA damaging agents to effectively reduce blastcells. These data show that, even in combination with dexamethasone,[Low]DXR does not act as a traditional chemotherapeutic while normal DXRdoes.

FIG. 12 shows single-dose DXR-loaded nanoparticles further reduce LSCsrelative to free DXR in chemotherapy treated leukemic mice. a-b,Leukemic mice established as described in FIG. 3a were treated with 5daily injections of free DXR at 0.5 or 0.15 pg/g with and withoutchemotherapy. Alternatively, a single injection on day 1 of 0.8 or 2.5pg/g of DXR-loaded nanoparticles (NanoDXR) was given with and withoutchemotherapy. At 10 days post-treatment, BM was analyzed by flowcytometry to determine frequency of LSCs (a) and HSPCs (b). Shown isaverage frequency±SD (n 6 per group). Note that 5 doses of 0.15 pg/g DXRis ineffective; however, a single NanoDXR injection with a similarcumulative dose (0.8 pg/g) is most effective at reducing LSCs whileallowing for HSPC recovery.

FIG. 13 illustrates that [Low]NanoDXR treatment reduces functional LSCsin vivo. a-b, Cohorts of leukemic mice were prepared and treated as inFIG. 3a but with [Low]NanoDXR. At 12 days post-treatment, BM washarvested from treated mice and transplanted into sub-lethallyirradiated NSG recipients. a, Treatment schematic and Kaplan-Meiercurves of recipient mice. The free [Low]DXR treatment group (solid line)from FIG. 5a is shown for comparison (n=30 per group). b, Recipients ofBM from [Low]DXR and [Low]NanoDXR treated leukemic mice were analyzed byflow cytometry at 6 months post-transplant for Blast cells, HSPCs andLSCs (n=27-29 per group).

FIG. 14 illustrates that doxorubicin nanoparticles have enhancedeffectiveness in eliminating leukemic stem cells and facilitating normalhematopoietic stem/progenitor cell recovery compared to Doxil®. Leukemicmice were injected with vehicle, chemotherapy, or chemotherapy combinedwith five daily low doses of doxorubicin (Doxo), nanoparticleencapsulated doxorubicin (NanoDoxo), or Doxil® (see Materials andMethods). Bone marrow was harvested 5-6 days after treatment andanalyzed by flow cytometry for HSPCs (identified as lineage negative,Sca-1⁺, c-Kit⁺ cells) and LSCs (lineage negative, c-Kit^(Mid), CD3⁺cells). LSCs and HSPCs were quantified by frequency (A, C) and absolutenumber per femur (B, D).

FIG. 15 provides a, in vivo circulation time of free doxorubicin (DOX)and doxorubicin nanoparticles (DOX-NPs). b, shows tissue distribution ofDOX and DOX-NPs in tumor-bearing SCID mice after 24 h injection. Dataare presented as mean±SD (n =5) *P<0.05, **P<0.01. c provides cardiacTroponin I level after administration of free doxorubicin (DOX) anddoxorubicin nanoparticles (DOX-NPs).

DETAILED DESCRIPTION OF THE INVENTION

Before the disclosed methods and materials are described, it is to beunderstood that the aspects described herein are not limited to specificembodiments, methods, apparati, or configurations, and as such can, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular aspects only and,unless specifically defined herein, is not intended to be limiting.

In view of the present disclosure, the methods described herein can beconfigured by the person of ordinary skill in the art to meet thedesired need. For example, in certain aspect, the disclosure providesmethods of treating cancer, comprising administering to a subject inneed thereof a pharmaceutically active molecule that is capable ofselectively inhibiting p-S⁵⁵²-β-catenin, p-T²¹⁷-β-catenin,p-T³³²-β-catenin, and/or p-S⁶⁷⁵-β-catenin production and/or activity,wherein the pharmaceutically active molecule is administered in anamount effective to reduce and/or limit cancer-initiating cells. Incertain embodiments, the cancer is resistant to traditional treatment.For example, the cancer of the disclosure is resistant to radiationtherapy, chemotherapy, immunotherapy, or any combination thereof.

In other embodiments, the cancer is selected from the group consistingof leukemia, lymphoma, prostate cancer, breast cancer, endometrialcancer, gastrointestinal cancer, lung cancer, melanoma, sarcoma,neuroblastoma, mesothelioma, testicular cancer, thyroid cancer, ovariancancer, uterine cancer, pancreatic cancer, liver cancer, and Wilms'Tumor. In one embodiment, the cancer is leukemia.

In certain embodiments, the pharmaceutically active molecule isadministered in a low dose and/or slow release and/or nanoparticleformulation. In one embodiment, the low dose is the dose at which theinhibitory effect of the pharmaceutically active molecule on knowntopoisomerase II-dependent cytotoxicity that requires higher dosage isreduced. For example, the low dose may be about ⅕ to about 1/50 of theclinical dose of the pharmaceutically active molecule when dosed forchemotherapy, wherein the clinical dose is the human dose approved foruse in any country.

In certain embodiments, the low dose may be about ⅕ to about 1/40, orabout ⅕ to about 1/30, or about ⅕ to about 1/25, or about ⅕ to about1/20, or about ⅕ to about 1/15, or about ⅕ to about 1/10, or about ⅙ toabout 1/50, or about 1/7 to about 1/50, or about 1/10 to about 1/50, orabout 1/15 to about 1/50, or about 1/20 to about 1/50, or about 1/25 toabout 1/50, or about 1/30 to about 1/50, or about 1/40 to about 1/50,1/10 to about 1/40, or about 1/10 to about 1/30, or about 1/10 to about1/25, or about 1/10 to about 1/20, or about 1/10 to about 1/15, 1/20 toabout 1/40, or about 1/20 to about 1/30, or about 1/20 to about 1/25, orabout 1/30 to about 1/40, or about 1/15 to about 1/25, or about ⅙ toabout 1/30, or about 1/7 to about 1/30, or about 1/10 to about 1/30, orabout 1/15 to about 1/30, or about 1/20 to about 1/30, or about 1/25 toabout 1/30, or up to about 1/50, or up to about ¼, or up to about 1/35,or up to about 1/30, or up to about 1/25, or up to about 1/20, or up toabout 1/15, or up to about 1/10, or up to about ⅛, or up to about ⅙, orup to about ⅕, the clinical dose of the pharmaceutically active moleculewhen dosed for chemotherapy, wherein the clinical dose is the human doseapproved for use in the U.S. or any other country.

In certain embodiments, the low dose may be about 0.01 to about 30mg/m²/day of the pharmaceutically active molecule. In other embodiments,the low dose is from about 0.01 to about 25, or about 0.01 to about 20,or about 0.01 to about 15, or about 0.01 to about 10, or about 0.01 toabout 9, or about 0.01 to about 7.5, or about 0.01 to about 5, or about0.01 to about 3, or about 0.01 to about 2, or about 0.1 to about 30, orabout 0.1 to about 25, or about 0.1 to about 20, or about 0.1 to about15, or about 0.1 to about 10, or about 0.1 to about 9, or about 0.1 toabout 7.5, or about 0.1 to about 5, or about 0.1 to about 3, or about0.1 to about 2, or about 1 to about 30, or about 1 to about 25, or about1 to about 20, or about 1 to about 15, or about 1 to about 10, or about1 to about 9, or about 1 to about 7.5, or about 1 to about 5, or about 1to about 3, or about 1 to about 2, or about 5 to about 30, or about 5 toabout 25, or about 5 to about 20, or about 5 to about 15, or about 5 toabout 10, or about 5 to about 9, or about 5 to about 7.5, or about 10 toabout 30, or about 10 to about 25, or about 10 to about 20, or about 10to about 15, or about 15 to about 30, or about 15 to about 25, or about15 to about 20, or about 20 to about 30, or up to about 0.01, or fromabout 0.01 and up to about 0.05, or up to about 0.1, or up to about 0.5,or up to about 1, or up to about 2, or up to about 3, or up to about 4,or up to about 5, or up to about 6, or up to about 7, or up to about 8,or up to about 9, or up to about 10, or up to about 15, or up to about20, or up to about 25, or up to 26, or up to 28, or up to 30, mg/m²/dayof the pharmaceutically active molecule. In non-limiting example,doxorubicin may be administered at a dose of about 7 to about 8, orabout 7.5 to about 8.5 mg/m²/day. In non-limiting example, doxorubicinmay be administered at a dose of up to about 10 mg/m²/day. Innon-limiting example, doxorubicin nanoparticles may be administered at adose of about 2 to about 3, or about 2.4 5 mg/m²/week (e.g., about 0.34mg/m²/day).

In certain embodiments, the low dose may be about 20 to about 50mg/m²/day of the pharmaceutically active molecule. In other embodiments,the low dose is from about 25 to about 50, or about 30 to about 50, orabout 40 to about 50, or about 20 to about 45, or about 20 to about 40,or about 25 to about 45, or up to about 50, or up to about 45, or up toabout 40, or up to about 35, mg/m²/day of the pharmaceutically activemolecule.

In certain embodiments, the pharmaceutically active molecule isanthracycline or a pharmaceutically acceptable salt thereof.Anthracyclines are a class of compounds derived from Streptomycesbacterium. Examples include, but are not limited to, doxorubicin,daunorubicin, epirubicin, idarubicin, valrubicin, and mitoxantrone. Inone embodiment, the pharmaceutically active molecule is doxorubicin ordaunorubicin. In another embodiment, the pharmaceutically activemolecule is daunorubicin or a pharmaceutically acceptable salt thereof.In yet another embodiment, the pharmaceutically active molecule isdoxorubicin or a pharmaceutically acceptable salt thereof.

In certain embodiments, the pharmaceutically active molecule ishydrophobic molecule.

In certain embodiments, the pharmaceutically active molecule is any oneof compositions disclosed in International Publication No. WO2015/054269 and International Publication No. WO 2016/061310, bothincorporated herein by reference in their entirety.

The methods of the disclosure, in one aspect, may compriseadministration of the pharmaceutically active molecule in a nanoparticleform (e.g., core/shell nanoparticle form). Such nanoparticles are ableto encapsulate large amount of hydrophobic drug molecules into thenanoparticles during the self-assembling process. The hydrophilicsurface protects the nanoparticles from reticuloendothelial system (RES)uptake and facilitates long circulation in body. Furthermore, thenanoparticles significantly increased the duration of the drug in thecirculation and decreased cardiac accumulation of the drug. Finally, thenanoparticles of the disclosure may be used for intracellular deliveryof anticancer drugs with minimal toxicity.

In some embodiments, wherein the pharmaceutically active molecule asdescribed above is administered in one or more nanoparticle compositionscomprising a block copolymer in a core/shell form, wherein the blockcopolymer comprises:

a first block, which is of formula:

and a second block, which is of formula:

wherein

m and n are independently an integer about 3 to about 500;

A is independently selected from polynorbonene, polycyclopentene,polycyclooctene, polyacrylate, polymethacrylate, a polysiloxane,polylactide, polycaprolactone, polyester, and polypeptide;

R₁ is a steroid moiety optionally comprising a linker; and

R₂ is a polyalkylene oxide moiety.

The block copolymers useful in the methods of the disclosure requirethat R₁ comprises a steroid moiety optionally comprising a linker. Asthe person of ordinary skill in the art will appreciate, suitablesteroids may be selected to meet the desired need. For example, thesteroid moiety suitable in the materials of the disclosure comprisescholesterol, cholic acid, deoxycholic acid, taurocholic acid,lanosterol, estradiol, testosterone, bile acid, dexamethasone,secosteroid, or phytosterol. In some embodiments, the steroid moietycomprises cholesterol, cholic acid, deoxycholic acid, taurocholic acidor the like. In one embodiment, the steroid moiety comprisescholesterol.

The steroid moiety may be connected to the polymer back bone via asuitable linker. Some examples of linkers include, but are not limitedto:

a polylactone, or an oligomer of siloxane. In one embodiment, the linkerat R₁ is:

In another embodiment, the linker at R₁ is:

The block copolymers useful in the methods of the disclosure requirethat R₂ comprises a polyalkylene oxide moiety. As the person of ordinaryskill in the art will appreciate, suitable polyalkylene oxides may beselected to meet the desired need. In some embodiments, the polyalkyleneoxide moiety comprises polyethylene oxide or polyethylene oxidethiolate. In another embodiment, the polyalkylene oxide moiety comprisespolyethylene oxide.

The block copolymers useful in the methods of the disclosure require abackbone moiety A. The block copolymers described herein may contain,for example, polynorbonene, polycyclopentene, polycyclooctene,polyacrylate, polymethacrylate, and a polysiloxane backbone A availableto one skill in the art, and may be varied depending on the desiredproduct. In one embodiment, the block copolymers of disclosure are thosewherein each A is independently polynorbonene or polyacrylate. Inanother embodiment, each A is independently polynorbonene. In anotherembodiment, each A is independently polyacrylate.

In one embodiment, the block copolymers useful in the methods of thedisclosure comprise the structure:

wherein x

is an integer between about 3 and about 100; m is an integer betweenabout 5 and about 200; and n is an integer between about 5 and about100. In some embodiments, x is between about 5 and 50. In otherembodiments, x is about 8, or x is about 44.

The values of m and n may be selected by one of skill in the art and maybe varied depending on the desired product. For example, m may bebetween about 10 and about 100; and/or n may be between about 15 andabout 85. The molecular weight of the block copolymer of the disclosuremay be between about 10,000 and about 1,000,000 Da. In one embodiment,the block copolymer of the disclosure is about 40,000 to about 750,000Da, or about 60,000 to about 700,000 Da, or about 60,000 to about100,000 Da, or about 40,000 to about 200,000 Da.

In some other embodiments, wherein the pharmaceutically active moleculeas described above is administered in one or more nanoparticlecompositions comprising a block copolymer in a core/shell form, whereinthe block copolymer comprises:

a first block, which is of formula:

and a second block, which is of formula:

wherein

q is an integer about 3 to about 500;

A¹ is independently selected from polyacrylate, polymethacrylate,polynorbonene, polycyclopentene, polycyclooctene, polysiloxane,polylactide, polycaprolactone,polyester, and polypeptide;

R¹¹ is a steroid moiety optionally comprising a linker R¹⁴;

R¹² polyalkylene oxide, polyester, or polypeptide moiety; and

R¹³ is a disulfide linker moiety.

In some embodiments, the steroid moiety in R¹¹ comprises cholesterol,cholic acid, deoxycholic acid, taurocholic acid, lanosterol, estradiol,testosterone, bile acid, dexamethasone, secosteroid, phytosterol, or thelike. In another embodiment, the steroid moiety in R¹¹ is selected fromcholesterol, cholic acid, deoxycholic acid, and taurocholic acid. Inanother embodiment, the steroid moiety in R¹¹ comprises cholesterol.

The steroid moiety may be connected to the polymer back bone via asuitable linker R¹⁴. Some examples of linker R¹⁴ include, but are notlimited to:

a polylactone, or an oligomer of siloxane. In one embodiment, the linkerat R¹⁴ is

. In another embodiment, the linker at R¹⁴ is

In another

embodiment, the linker at R¹⁴ is In one embodiment, the

linker at R¹⁴ is

In one embodiment, the block copolymers useful in the methods ofdisclosure are those wherein each A¹ is independently polyacrylate,polymethacrylate, or polyester. In another embodiment, each A isindependently polyacrylate or polymethacrylate. In another embodiment,each A¹ is independently polyacrylate. In another embodiment, each A¹ isindependently polymethacrylate. In another embodiment, each A¹ isindependently polyester.

In an exemplary embodiment, the first block is of formula:

In one embodiment, R¹² is polyalkylene oxide moiety. Suitablepolyalkylene oxides may be selected to meet the desired need. In someembodiments, the polyalkylene oxide moiety comprises polyethylene oxide,polyethylene oxide thiolate, polypropylene oxide, or polypropylene oxidethiolate. In another embodiment, the polyalkylene oxide moiety comprisespolyethylene oxide or polyethylene oxide thiolate. In anotherembodiment, the polyalkylene oxide moiety comprises polyethylene oxide.

In one embodiment, R¹² is polyester moiety. Suitable polyesters includepolymers that contain the ester functional group in their main chain.Examples include, but are not limited to, polylactides, polyglycolides,polycaprolactones, and the like.

In one embodiment, R¹² is polypeptide moiety. Suitable polypeptidesinclude one or more chains of amino acid monomers linked together bypeptide (amide) bonds, and may comprise L-amino acids, D-amino acids(which are resistant to L-amino acid-specific proteases in vivo), or acombination of D- and L-amino acids. Typically, polypeptides describedherein refer to a chain less than about 100 amino acids in length. Thepolypeptides described herein may be chemically synthesized orrecombinantly expressed.

The second block also comprises R¹³ linker moiety comprising reducibledisulfide bonds. In one embodiment, R³ is selected from the groupconsisting of:

In one embodiment, R¹³ is

In another embodiment, R¹³ is derived from

In certain embodiment, the copolymer of the disclosure may furthercomprise a chain terminus moiety X:

In one embodiment, X is a trithiocarbonate, dithiocarbamate, ordithioester. In another embodiment, X is —SC(S)S—(C₁-C₂₄ alkyl). Inanother embodiment, X is —SC(S)S—(C₁₂H₂₅.

In one embodiment, the block copolymers useful in the methods of thedisclosure comprise the structure:

wherein q is an integer between about 5 and about 200; and r is aninteger between about 5 and about 100.

The values of q and r may be selected by one of skill in the art and maybe varied depending on the desired product. For example, q may bebetween about 10 and about 100; and/or r may be between about 15 andabout 85. The molecular weight of the block copolymer of the disclosuremay be between about 5,000 to about 200,000 Da. In one embodiment, theblock copolymer of the disclosure is about 5,000 to about 150,000 Da, orabout 5,000 to about 100,000 Da, about 5,000 to about 60,000 Da, orabout 10,000 to about 150,000 Da, or about 10,000 to about 100,000 Da,or about 10,000 to about 60,000 Da, or about 20,000 to about 150,000 Da,or about 20,000 to about 100,000 Da, or about 20,000 to about 60,000 Da.

In one embodiment, the methods of the disclosure comprise administrationof the pharmaceutically active molecule in a combination of twodifferent nanoparticle compositions. In some embodiments, a firstnanoparticle composition comprises the hydrophobic pharmaceuticallyactive molecule that is doxorubicin. In other embodiments, a secondnanoparticle composition comprises the hydrophobic pharmaceuticallyactive molecule selected from the group consisting of daunorubicin,vincristine, epirubicin, idarubicin, valrubicin, mitoxantrone,paclitaxel, docetaxel, cisplatin, camptothecin, irinotecan,5-fluorouracil, methotrexate, or dexamethasone. In some otherembodiments, a second nanoparticle composition comprises the hydrophobicpharmaceutically active molecule selected from daunorubicin andepirubicin.

The nanoparticles useful in the methods of the disclosure may furthercomprise one or more of metal nanoparticles, such as gold nanoparticlesand/or magnetic nanoparticles and/or quantum dots (for example, nearinfrared (NIR) quantum dot, CdSe and the like).

The nanoparticles useful in the methods of the disclosure of thedisclosure may be anywhere from about 5 to about 900 nm in size. Forexample, the nanoparticles may be between about 5 and about 200 nm, orbetween about 10 and about 100 nm, or between about 10 and about 200 nm,or between about 50 and about 150 nm, or between about 100 and about 250nm, or between about 100 and about 200 nm, or between about 120 andabout 150 nm, or between about 110 and about 150 nm, or between about120 and about 180 nm, or between about 150 and about 250 nm, or betweenabout 150 and about 200 nm.

Definitions

Throughout this specification, unless the context requires otherwise,the word “comprise” and “include” and variations (e.g., “comprises,”“comprising,” “includes,” “including”) will be understood to imply theinclusion of a stated component, feature, element, or step or group ofcomponents, features, elements or steps but not the exclusion of anyother integer or step or group of integers or steps.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. In some embodiments, the term “about” means ±10% of therecited value. In another embodiment, term “about” means ±5% of therecited value.

The term “activity,” for example of a protein, as used herein includesdirect activity of that protein and indirect downstream activity of thatprotein. For example, interaction with the protein 14-β-3zeta results instabilization of β-catenin, enhanced nuclear localization of β-catenin,enhanced binding/activity to TCF/LEF transcription factor sites, oractivation of a molecular program resulting in stem cell proliferation.

The term “cancer-initiating cells” (CICs), e.g., “cancer stem cells”(CSCs), as used herein include cells that have the ability to generateor regenerate tumors. In certain embodiments CICs are resistant tostandard chemotherapeutic treatment.

As used herein the term “combining” includes adding one or more items toa reaction mixture.

As used herein the term “dispersity,” “polydispersity,” “polydispersityindex”, “PDI,” and “M_(w)/M_(n)” are used interchangeably and refer tomeasure of the polymer uniformity with respect to distribution ofmolecular mass. The dispersity may be calculated by dividing weightaverage molecular weight (M_(w)) by the number average molecular weight(M_(n)) (i.e., M_(w)/M_(n)). In certain embodiments, the dispersity maybe calculated according to degree of polymerization, where thedispersity equals X_(w)/X_(n), where X, is the weight-average degree ofpolymerization and X_(n) is the number-average degree of polymerization.

All percentages, ratios and proportions herein are by weight, unlessotherwise specified. A weight percent (weight %, also as wt %) of acomponent, unless specifically stated to the contrary, is based on thetotal weight of the composition in which the component is included(e.g., on the total amount of the reaction mixture).

The terms “reduce and/or limit cancer-initiating cells” includes anyamount of absolute reduction (about 5%, about 10%, about 25%, about 50%,about 75%, about 95%, or greater, or complete elimination) ofcancer-initiating cells, and any amount of limiting the rate ofexpansion about 5%, about 10%, about 25%, about 50%, about 75%, about95%, or greater) as compared to cells receiving no treatment.

“Therapeutically effective amount” refers to that amount of a compoundwhich, when administered to a subject, is sufficient to effect treatmentfor a disease or disorder described herein. The amount of a compoundwhich constitutes a “therapeutically effective amount” will varydepending on the compound, the disorder and its severity, and the age ofthe subject to be treated, but can be determined routinely by one ofordinary skill in the art.

“Treating” or “treatment” as used herein covers the treatment of adisease or disorder described herein, in a subject, preferably a human,and includes:

i. inhibiting a disease or disorder, i.e., arresting its development;

ii. relieving a disease or disorder, i.e., causing regression of thedisorder;

iii. slowing progression of the disorder; and/or

iv. inhibiting, relieving, or slowing progression of one or moresymptoms of the disease or disorder.

“Subject” refers to a warm blooded animal such as a mammal, preferably ahuman, or a human child, which is afflicted with, or has the potentialto be afflicted with one or more diseases and disorders describedherein.

“Pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problems or complications commensurate with a reasonablebenefit/risk ratio or which have otherwise been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” refers to both acid and base additionsalts. The term “polyester” as used herein includes polymers thatcontain the ester functional group in their main chain. Non-limitingexamples include polylactides, polyglycolides, polycaprolactones, andthe like. The term “polypeptide” as used herein includes a chain ofamino acid monomers linked together by peptide (amide) bonds, and maycomprise L-amino acids, D-amino acids (which are resistant to L-aminoacid-specific proteases in vivo), or a combination of D- and L-aminoacids. Typically, polypeptides described herein refer to a chain lessthan about 100 amino acids in length. The polypeptides described hereinmay be chemically synthesized or recombinantly expressed.

EXAMPLES

The materials and methods of the disclosure are illustrated further bythe following examples, which are not to be construed as limiting thedisclosure in scope or spirit to the specific procedures and materialsdescribed in them.

Materials and Methods

Animals Mice were housed in the animal facility at Stowers Institute forMedical Research (SIMR) and handled according to Institute and NIHguidelines. All procedures were approved by the IACUC of SIMR. TheHSC-SCL-Cre-ER^(T) Pten^(IonP/IoxP βcat(Ctnnb)1)^(IonP(Expm3)/+)(hereafter, Pten:β-cat^(Act)) mouse model combines conditional deletionof LoxP flanked Pten, resulting in activation of the PI3K/Akt pathway,and exon 3 of β-catenin (β-cat^(Act)), resulting in constitutiveactivation of β-catenin. The hematopoietic stem/progenitor cells(HSPCs)-specific Cre recombinase, HSCSCL-Cre-ER^(T), was used to studyof the combined effects of both pathways starting with HSPCs and withoutthe HSC activating effects of induction by interferon. PrimaryHSC-SCL-Cre mice were induced by intra-peritoneal injection of tamoxifendaily for 5 days using 5 mg on day 1 and 2 mg on days 2-5 each dissolvedin 0.1 ml of corn oil. A Bioruptor® sonicator was used to fullysolubilize the tamoxifen. HSC-SCL-Cre was induced in transplantrecipients by placing transplant recipients on tamoxifen feed (1 mg/g)for 2 weeks. HSC-SCL-Cre, Pten, and β-cat^(Act), were obtained fromJoachim Goethert (University of Duisburg-Essen, Germany), Hong Wu (UCLA,Los Angeles, Calif.), and Makoto Taketo (Kyoto University, Japan),respectively.

Transplantation Assays

Whole bone marrow was isolated from uninduced HSC-SCL-Cre⁺Pten^(fx/fx)βcat^(fx(Exon3)/+) (Pten:β-cat^(Act)) mice and combined with an equalportion of Cre negative bone marrow from a littermate and transplantedinto irradiated (10 Gy) B6.SJL-Ptprc^(a) Pepc^(b)/BoyJ (Ptprc)recipients. Recipients were placed on Tamoxifen feed 4-6 weekspost-transplant to induce recombination, resulting in leukemiadevelopment by 7-8 weeks post-induction in all recipient mice.

Limiting-dilution and tumorigenic assays were performed by establishingleukemic mice as described above and treating as indicated at 8 weekspost-induction. For limiting-dilution transplants, mice were treatedwith chemotherapy or [Low]DXR and, at 10 days post-treatment (based onfirst treatment), CD45^(Hi) CD³⁺ c-Kit⁻ blast cells or Lin⁻ CD³⁺c-Kit^(Mid) LSCs were sorted from chemotherapy treated mice andLin⁻Sca-1⁺ c-Kit⁺HSPCs were sorted from [Low]DXR treated mice. Theindicated numbers of these populations were transplanted into 3.25 Gyirradiated NOD.Cg-Prkdc^(scid) II2rg^(tm1Wjl)/SzJ (NSG) recipient mice.Recipient bone marrow was analyzed by flow cytometry at 10-12 weekspost-transplant and those with ≥1% CD45^(Hi) blast cells in bone marrowwere considered engrafted. CRU frequency was determined using ELDAanalysis.

Tumorigenic assays were performed by transplanting 0.5, 1.5, or 4.5×10⁴bone marrow cells from treated mice at 12 days post-treatment into 3.25Gy irradiated NSG recipient mice. 10 recipients were used for each dosefrom each group. One male and one female donor was used for each group.Leukemia was assessed in mice euthanized due to poor health by analyzingCD45^(Hi) CD3⁺ cell frequency. Mice having >20% Blasts in the bonemarrow were considered leukemic. NSG and Ptprc mice were originallyobtained from The Jackson Laboratory.

In vitro Treatment

Bone marrow cells from leukemic mice at 8 weeks post-induction werecultured overnight at 5−20×10⁴ cells per well in 96-well U-bottom tissueculture plates (Becton, Dickinson and Company; Cat. No. 353077) in HSCexpansion media in low O₂ conditions as previously described (Perry, J.M. et al. Cooperation between both Wnt/{beta}-catenin and PTEN/PI3K/Aktsignaling promotes primitive hematopoietic stem cell self-renewal andexpansion. Genes Dev 25, 1928-1942 (2011)). Doxorubicin (Sigma; D1515),0105375((S)-N-(2-bromobenzyl)-N-(1-hydroxy-β-phenylpropan-2-yl)ethenesulfonamide,University of Kansas CMLD compound), or Thioguanosine was mixed with HSCexpansion media and added to the cultures to obtain final concentrationsof 11, 33, 100 nM. Equivalent amounts of DMSO alone (vehicle control)were added to parallel cultures for comparison. Half-media changes wereperformed approximately every 24 hrs. Cultures were analyzed after 72hrs exposure to the indicated drug.

In vivo Treatment

Chemotherapy consisted of Nelarabine (Selleck) and Dexamethasone(BioVision) administered daily for 5 days consecutively. 43.4 mg/mlNelarabine was administered intravenously via the tail vain according tothe formula: Body Weight (g)×5=volume to inject (p1), which yielded 217mg/kg. 2.5 mg/ml Dexamethasone was injected intraperitoneally accordingto the formula: Body Weight (g)×4=volume to inject (μl), yielding 10mg/kg. [Low]DXR (also referred to as [Low]Doxo or throughout) treatmentconsisted of 5 consecutive daily doses at 0.5 mg/kg using Doxorubicinhydrochloride (Sigma; D1515) at 0.1 mg/ml injected intravenously via thetail vain according to the formula: Body Weight (g)×5=volume to inject(p1), which yielded 0.5 mg/kg. [Low]NanoDXR (also referred to as[Low]NanoDoxo throughout) treatment used doxorubicin nanoparticles asdescribed in International Patent Publication WO 2015/054269(incorporated by reference in its entirety) administered as a single IVinjection once per week on day 1 relative to above treatments using 0.8mg/kg. Maintenance [Low]NanoDXR consisted of once per week injections of0.4 mg/kg. Groups combining Nelarabine with Doxorubicin used a singleinjection containing both drugs. All drugs were solubilized in 45%(2-Hydroxypropyl)-β-cyclodextrin (HBC).

Rationale for doxorubicin dosage: for clinical ALL therapy, doxorubicinis typically administered at a single dose every 21-28 days at 40-75mg/m². Using 60 mg/m² as the clinical equivalent dose, this isequivalent to 1.6 mg/kg for adult humans (60 mg/m^(2×1) m²/37 kg=1.6mg/kg). Converting to mouse, this is equivalent to −20 mg/kg (1.6mg/kg×12.3 (k_(m(Human))/k_(m(Mouse)))=19.7 mg/kg) (Freireich, E. J. etal., Quantitative comparison of toxicity of anticancer agents in mouse,rat, hamster, dog, monkey, and man. Cancer chemotherapy reports. Part 150, 219-244 (1966).). Cumulatively, 2.5 mg/kg doxorubicin wasadministered and thus 1/8 the equivalent clinical dose spread over 5days.

Flow Cytometry

Cells were collected from bone marrow (femur and tibia), spleen,peripheral blood, and thymus. For cell surface phenotyping, a lineagecocktail (Lin) was used including CD3 (for HSPC but not LSC analysis),CD4, CD8, Mac-1, Gr1, B220, IgM, and Ter119 (eBioscience, San Diego,Calif.). Monoclonal antibodies against CD3 (separate fluorophore for LSCanalysis), Sca-1, c-Kit, CD45.1, and CD45.2 were also used whereindicated. Cell sorting and analysis were performed using an inFlux(BD), MoFlo (Dako, Ft. Collins, CO) and/or CyAn ADP (Dako, Ft. Collins,CO). Data analysis was performed using FlowJo software (Ashland, OR).

Immunostaining

Cells were sorted onto lysine-coated slides, fixed with methanol,blocked using Universal Block, and stained for pS552-β-catenin at 1:50dilution.

FRET assay

FRET measurement was performed by using the acceptor photobleachingmethod. Briefly, 293T cells were transfected with EGFP-AKT andmCherry-β-catenin (Addgene, #39531, #55001). A Perkin-Elmer Ultraviewspinning disc system with a CSU-X1 Yokogawa disc was used for imaging. A40X 1.2 NA Plan-apochromatic objective was used, and emission wascollected onto a C9100 Hamamatsu Photonics EM-CCD. EGFP was excited witha 488 nm laser, and emission was collected through a 500-555 nm bandpass filter. mCherry was illuminated, and photobleached, with a 561 nmlaser. Emission of mCherry was collected with a 580-650 nm band passfilter. 6 images of EGFP were acquired before and 8 images afterbleaching of the mCherry with intense 561 nm light. After subtraction ofcamera background, the average intensity of EGFP in a region of interestspanning the bleached cell was determined in the 4 images beforeacceptor bleach (11), or the 4 images after acceptor bleach (12). FRETefficiency is reported as 1-(11/12). Calculations were based on >500cell images.

High-Throughput Screening

243 compounds were selected from primary screening of the validationlibrary (5040 compounds) drawn from CMLD (1920), Prestwick (1120) andMicroSource Spectrum (2000) and reconfirmed in a 10 concentrationdose-response. Activity of compounds was tested against HEK-TOP cellsvs. HEK FOP cells for inhibition of luciferase activity. Thecytotoxicity profiles of the compounds were also tested using Cell TiterGlo assay (Promega) on HEK-TOP cell lines. The dose-response data wasused to calculate the EC50 (Effective concentration of compoundsresulting in 50% inhibition of luminescence or cytotoxicity) usingnonlinear regression analysis. Approximately 90 compounds showed from2.2 to 3 fold differences in EC50 between the TOP and FOP cells. Ofthese 36 compounds showed a window between luminescence inhibition andcytotoxicity. The structures of compounds were analyzed bycheminformatics analysis and medicinal chemists identified 25 compoundsfor repurchasing as fresh powders. The repurchased compounds were usedto treat the cells at compound concentrations that resulted in 90% , 50%and 25% inhibition of luminescence(EC50, EC50 and EC25), derived fromthe dose-response curves for luminescence inhibition in HEK Top cellline. The HEK cells and HEK Top cells were plated at 300,000 cells/wellin 6 well plates and were treated in duplicate with EC90, EC50 and EC25concentrations of the 25 repurchased compounds as well as threecontrols. After 48h of exposure, the cells were washed with PBS andflash frozen. The frozen cells were lysed directly in plates for Westernanalysis.

Statistical Analyses

Data expressed as mean±standard deviation. Pair-wise comparisonsperformed using Student's t-test.

Example 1 Simultaneous Activation of Wnt/β-catenin and PI3K/Akt pathwaysresults in Successive Expansion of HSPCs, LSCs and T-ALL Blast Cells

Previous work showed that cooperative activation of the Wnt/β-cateninand PI3K/Akt pathways drove self-renewal but resulted in leukemictransformation. The ontogeny and nature of leukemogenesis inPten:β-cat^(Act) mice that activate both pathways in HSPCs was explored.Pten:β-cat^(Act) double mutants consistently and robustly developedblast crisis as indicated by >20% CD45^(hi) leukemic blasts. These cellswere negative for major lineage markers but expressed CD3 while beingnegative for CD4 and CD8, suggesting a maturation blockage in earlyT-cell development (FIG. 1a-b ). Analysis of major lineages from BM,spleen, and thymus showed a reduction in mature lineages with a largeincrease in immature (double negative) thymocytes in the thymus and withCD3⁺ blasts overtaking the BM compartment by 9-10 weeks post-induction(wpi) (FIG. 6). To trace the ontogeny of this T-cell acute lymphocyticleukemia (T-ALL), we analyzed bone marrow at earlier time points forHSPCs and LSCs, the former identified as lineage negative (Lin),Sca-1⁺c-Kit⁺ cells and the latter as CD3⁺ (but otherwise Lin⁻) andc-Kit^(Mid) cells²⁰. Similar to our previous study, we found a strikingaccumulation of HSPCs in double mutants at 6 wpi with commensuratereduction in more mature (Lin⁻c-Kit⁺Sca-1⁻) progenitor cells consistentwith broad differentiation blockage (FIG. 1c ; see Perry et al. 2011 forfurther details on HSC expansion). At this stage we also found a rarepopulation of LSCs, which became more frequent by 8 wpi as the HSPCpopulation collapsed (FIG. 1d ). By 12 wpi, all double mutant micesuccumbed to T-ALL (FIG. 1e ). This rapid and consistent T-ALLdevelopment was unique to double mutants. Only 25% of Pten singlemutants developed leukemia within 6 months post-induction.Histologically, the marrow cavity was largely taken over by leukemicblasts, but the trabecular bone region—the main site of the HSCniche—maintained a residual holdout of hematopoiesis (FIG. 7a ). Doublemutants also exhibited splenomegaly and disruption of the normalarchitecture (FIG. 7b -c). Interestingly, pS⁵⁵²-β-catenin positive cellswere particularly increased in double mutant spleens (FIG. 1f ).Collectively, these data demonstrate that cooperative activation of theWnt/β-catenin and PI3K/Akt pathways drive the progressive expansion ofphenotypic HSPCs with transformation to LSCs resulting in T-ALL.

EXAMPLE 2 DXR Targets pS⁵⁵²-β-Catenin while Sparing Total β-Catenin

Since pharmacological activation of the Wnt/β-catenin and PI3K/Aktpathways in normal HSCs synergistically drives self-renewal andexpansion, it was tested whether inhibition of this cooperation couldprevent oncogenic self-renewal. While there was some success in reducingLSCs using a novel pan-β-catenin inhibitor, the focus was on inhibitingthe pS⁵⁵²-β-catenin active form of β-catenin to target the cooperativeactivity of the pathways more specifically and to have lower toxicitythan a pan-β-catenin inhibitor (FIG. 8). High-throughput screening (HTS)identified several candidates for this specific inhibition (FIG. 2a ),which was narrowed to 3 compounds based on their ability to inhibitpS⁵⁵²-β-catenin with less effect on pan β-catenin: DXR, 0105375, andthioguanosine (FIG. 2b-c ). Interestingly, DXR and thioguanosine areknown anti-cancer agents, with DXR being among the most successfulanti-cancer therapies and thioguanosine being used originally for ALLand subsequently other tumors. The novel compound 0105375 effectivelyinhibited pS⁵⁵²-β-catenin with no significant effect on pan β-catenin,but only at relatively high concentrations (>1 μM) (FIG. 2c ). Althoughthioguanosine inhibited pS⁵⁵²-β-catenin while largely sparing panβ-catenin, it had high levels of overall toxicity (FIG. 2b-c ). Mostpromising appeared to be DXR, which effectively inhibitedpS⁵⁵²-β-catenin while have only minimal effect on pan β-catenin atrelatively low concentrations (FIG. 2b-c ). Indeed, computationalmodeling indicated that both Akt and DXR bound near the ^(pS552) site onβ-catenin (FIG. 2d ).

To test whether DXR could block interaction between Akt and β-catenin,Fluorescence Resonance Energy Transfer (FRET) analysis using EGFP-AKTand mCherry-β-catenin transfected cells was performed. FRET efficiencywas 2.24% in vehicle treated cells but decreased with increasingconcentrations of DXR (FIG. 2e ) and exposure time to DXR (FIG. 2f ).However, cells transfected with EGFP only and mCherry-β-catenin showedno discernible FRET and no difference in vehicle vs. DXR treatment (FIG.9). These data demonstrate that Akt interacts with β-catenin and thatDXR effectively inhibits this interaction.

The effects of these candidate drugs on BM cells isolated from leukemicdouble mutants were tested in vitro. Relative to vehicle, DXRsignificantly reduced LSCs but not HSPCs (FIG. 10). Since thioguanosinereduced not only LSCs but also HSPCs and since 0105375 had far lesspotency than DXR, we focused our in vivo studies on DXR. Together, thesedata show that DXR can inhibit p5⁵⁵²-β-catenin with minimal effects ontotal β-catenin. Interestingly, DXR exhibits the broadest spectrum ofanti-cancer activity known and has been employed as a standardchemotherapeutic agent for decades, but severe side effects limit itsuse. Toxicity, however, may be reduced if DXR were repurposed as atargeted PS⁵⁵²-β-catenin inhibitor rather than a DNA damaging agent.

EXAMPLE 3 Low-Dose DXR Treatment Inhibits the High Level ofpS552-β-Catenin Uniquely Overexpressed in Chemoresistant LSCs.

It was next determined whether DXR could inhibit pS⁵⁵²-β-catenin invivo. To obtain a relatively large set of leukemic mice that could beconsistently used at the same stage after induction to test differenttreatment regimens, whole BM from uninduced Pten:β-cat^(Act) mice wastransplanted into irradiated recipients. This was combined at a 1:1ratio with Cre negative BM from littermates to allow for potentialcompetition between normal and leukemic cells and to more closelyreflect clinical circumstances. After 4-6 weeks for recovery andengraftment, mice were placed on tamoxifen feed to induce recombination.After 8 weeks, leukemia was established in all recipients, which thenreceived various treatments (FIG. 3a ).

To repurpose DXR as a targeted inhibitor of pS⁵⁵²-β-catenin rather thana cytotoxic chemotherapeutic drug, doses well below the typical clinicaldose were used. While DXR is typically given as a bolus injection onceevery 3-4 weeks, 1/40^(th) the clinical equivalent dose (0.5 μg/g) wasadministered daily for 5 consecutive days, yielding a cumulative dose of118^(th) the typical amount (termed [Low]DXR) (FIG. 3b ). To determineif DXR could inhibit pS⁵⁵²-β-catenin and in which cells, HSPCs, LSCs,and non-LSC blast cells were sorted from treated mice using flowcytometry and stained for pS⁵⁵²-β-catenin (FIG. 3c ). WhilepS⁵⁵²-β-catenin was near background levels in blast cells and HSPCs,LSCs had an elevated average of pS⁵⁵²-β-catenin. This average wasreduced in LSCs isolated from mice recently treated with [Low]DXR,although consistent variability yielded no statistical significance inthis difference (FIG. 3d-e ).

To see how HSPCs, LSCs and blast cells responded to chemotherapy and howthis might affect their pS⁵⁵²-β-catenin status, additional groups weretreated with chemotherapy alone (see Methods) or chemotherapy with[Low]DXR. Interestingly, LSCs, but neither HSPCs nor blast cells,consistently expressed significantly high levels of pS⁵⁵²-β-catenin inresponse to chemotherapy. Notably, combined with [Low]DXR treatment,pS⁵⁵²-β-catenin levels were significantly reduced to background levels(FIG. 3d -e). These data demonstrate first that chemotherapy inducesconsistently high levels of pS⁵⁵²-β-catenin specifically and uniquely inLSCs, and second, that DXR can be repurposed using a low-dose to serveas an inhibitor of pS⁵⁵²-β-catenin.

Example 4 Targeting pS552-β-Catenin Selectively EliminatesChemoresistant LSCs.

Next the differential effects of chemotherapy and [Low]DXR on blastcells, LSCs, and HSPCs was determined. As expected, chemotherapysubstantially reduced blast cells compared to vehicle. However, it alsoinduced a large expansion in LSCs but no significant change in HSPCs.Notably, [Low]DXR did not significantly reduce blast cells (FIG. 4a-d ),but the clinical equivalent dose of DXR acted similar to chemotherapy intheir reduction (FIG. 11), supporting a more specific, less cytotoxicrole for [Low]DXR treatment. However, [Low]DXR significantly reducedLSCs compared to vehicle and allowed significant recovery of HSPCs.Overall, chemotherapy and [Low]DXR displayed dichotomous effects onthese populations, with chemotherapy targeting blast cells but inducingLSC expansion, whereas [Low]DXR did not significantly target blasts butreduced LSCs. In combination, chemotherapy with [Low]DXR treatment notonly reduced blasts and prevented LSC expansion, but also essentiallyeliminated detectible LSCs. Combination treatment also allowed forconsistent and significant recovery of HSPCs (FIG. 4a-d ).

To quantify the tumorigenic activity of LSCs relative to blast cells inchemotherapy treated mice, limiting-dilution transplants was performedinto sub-lethally irradiated NOD-SKID-II2re (NSG) recipients. LSCssorted from chemotherapy treated mice exhibited a >1300-fold increase incompetitive-repopulating unit (CRU) activity compared to blast cellssorted from the same donors (FIG. 4e ; Table 1). LSCs sorted fromchemotherapy treated mice also had a 10-fold higher CRU frequency thanHSPCs sorted from [Low]DXR treated mice, and, unlike the LSCs and blastcells, these HSPCs only engrafted at low levels without developing intoleukemia by 10-12 weeks post-transplant (FIG. 4e ; Table 1). Together,FIGS. 3 and 4 show that chemotherapy not only fails to eliminate LSCsbut also induces expression of pS⁵⁵²-β-catenin and functional LSCexpansion. However, [Low]DXR treatment inhibits pS⁵⁵²-β-cateninexpression and LSC expansion. Notably, these treatments exhibitdifferential effects on LSCs and blast cells.

TABLE 1 Treatment Transplant of Donors Population Dose Tested EngraftedEstimate Lower Upper Chemo. Blast Cells 25,000 10 1 1/198,165 1/528,9061/74,247 75,000 9 3 Chemo. LSCs 50 10 2 1/149    1/289    1/77    150 107 [Low]DXR HSPCs 50 5 0 1/1,544  1/6,240  1/383   150 6 1 450 5 1

Example 5 Binary Targeting of Bulk Leukemic Blasts and ChemoresistantLSCs Substantially increases Long-Term Survival

It was next determined whether LSCs are not only phenotypically but alsofunctionally reduced by [Low]DXR treatment. A cohort of mice wasestablished as in FIG. 3a . One week after completion of treatment withvehicle, chemotherapy, [Low]DXR, or chemotherapy +[Low]DXR, wetransplanted BM from treated mice into irradiated NSG recipients to testfor tumorigenic cells. Recipients treated with vehicle succumbed toleukemia in a similar manner to primary mutants following induction(FIG. 5a ). However, recipients of BM from leukemic mice treated withchemotherapy succumbed more rapidly, although 25% of the group exhibitedprolonged survival. The reduced survival of most recipients wasconsistent with LSC expansion induced by chemotherapy (FIG. 4). Nearlyall (29/30) recipients of BM from mice treated with [Low]DXR aloneremained healthy nearly 6 months post-transplant. Analysis of these 29survivors revealed only trace levels of blast cells and LSCs, and HSPCswere within normal parameters (FIG. 5b ). These data support thespecific targeting of functional LSCs by [Low]DXR treatment.

Also most recipients of BM from chemotherapy +[Low]DXR treated micesuccumbed to leukemia by 6 months post-transplant; however, their mediansurvival was significantly extended from 44.5 days to 104.5 dayscompared to the chemotherapy alone group (FIG. 5a ). Thus, althoughcombination treatment essentially eliminates phenotypic LSCs, functionalLSCs ultimately recover with exposure to chemotherapy. Even so, [Low]DXRtreatment significantly reduces chemoresistant cells with LSC activity.

To determine long-term survival, cohorts of leukemic mice wereestablished as in FIG. 3a but this time observed treated mice long-termwithout transplantation into NSG mice. While mice treated with vehiclealone succumbed to leukemia at a similar rate as untreated mice, thosetreated with chemotherapy alone exhibited somewhat improved overallsurvival; nonetheless, all still succumbed by 50 days post-treatment(FIG. 5c ). [Low]DXR only treated mice exhibited only minor andinsignificant improvement, likely due to the minimal and insignificanteffect of [Low]DXR treatment on blast cells (FIG. 4a ). However,combining chemotherapy and [Low]DXR treatment significantly increasedsurvival compared to chemotherapy (p<0.05) or [Low]DXR (p<0.01) alone(FIG. 5c ).

Although significant, these improvements were incremental in the primarytreated mice (FIG. 5c ). Since functional LSCs were still presentfollowing combination (chemotherapy+[Low]DXR) treatment (FIG. 5a ),whiteout being bound to a particular theory, it was hypothesized thatusing only a single cycle of chemotherapy combined with a moresustained, maintenance treatment of [Low]DXR might better prevent LSCsfrom repopulating the leukemia. The potential tissue damage caused bydoxorubicin, especially when giving multiple injections, makes long-termmaintenance dosing of DXR impractical. DXR-loaded nanoparticles(nanoDXR) were then used not only to reduce potential tissue damage butalso to allow for a slow, more sustained release of DXR duringchemotherapy treatment. Because these nanoparticles provide steady,sustained release of DXR, using nanoDXR allows to alter the dosingschedule to avoid repeated, daily DXR injections. Multiple doses of freeDXR and nanoDXR were tested to further optimize this treatment (FIG.12). A ⅛^(th) clinical equivalent dose of nanoDXR delivered as a singleinjection on day 1 was as effective in reducing LSCs as the equivalentcumulative dose of free DXR distributed over 5 days (see Suppl.Methods). Moreover, this single injection could be reduced to a1/25^(th) clinical equivalent with at least the same efficacy (FIG. 12).Thus, a cohort of leukemic mice was tested as before with chemotherapycombined with only a single 1/25^(th) injection of nanoDXR on day 1.Since high levels of pS⁵⁵²-β-catenin were apparent only in response tochemotherapy, subsequent, weekly injections of nanoDXR were furtherreduced to 1/50^(th) for an additional 9 weeks of maintenance treatment.This regimen significantly reduced LSCs while also facilitatingsignificant recovery of HSPCs compared to free [Low]DXR (FIG. 5d ).Notably, median survival was extended to 139 days, with most micesuccumbing only after cessation of maintenance [Low]nanoDXR (FIG. 5e ).Mice surviving over 7 months post-chemotherapy treatment showed, atmost, only trace levels of blast cells and LSCs with normal levels ofHSPCs (FIG. 5f ). Tumorigenic LSCs were tested by transplanting bonemarrow from [Low]nanoDXR treated mice into NSG recipients as in FIG. 5a. Recipients showed similar survival to recipients of free [Low]DXRtreated mice and even lower numbers of LSCs (FIG. 13). Together, thesedata show first that functional LSCs are differentially targeted bychemotherapy and [Low]DXR—with chemotherapy activating LSCs while[Low]DXR inhibits LSCs in tumorigenic assays (FIG. 5a-b ). Second, thatcombination therapy is necessary to substantially improve survival inleukemic mice as chemotherapy eliminates blast cells while [Low]DXRreduces LSC frequency and prevents the resultant chemoresistant LSCexpansion (FIG. 5c ). And lastly, that chemotherapy combined withmaintenance treatment using [Low]nanoDXR substantially improveslong-term survival (FIG. 5e ).

Discussion

Tumorigenic cells can not only resist standard chemotherapy, but alsoactually expand in response to it, which clarifies why anti-cancertherapy often fails. Targeting CSCs/LSCs, ideally with minimal effect onnormal stem/progenitor cells, is crucial to future success.Unexpectedly, this disclosure provides that a long-used chemotherapeuticagent could be repurposed to this end. DXR acts as a topoisomerase IIinhibitor at high concentrations and exhibits the broadest spectrum ofanti-cancer activity known, but it's not clear why DXR would havegreater efficacy in many cancers than other topoisomerase II inhibitors.However, anti-cancer drugs used as DNA damaging agents can haveunanticipated effects. Topotecan, another topoisomerase II inhibitor,was found to unsilence the normally dormant paternal allele of the Ube3agene when used at relatively low doses. Ube3a is responsible forAngelman syndrome when the maternal allele is mutated, so epigeneticde-repression of the typically normal paternal allele may alleviate thissyndrome. DXR is also known to affect epigenetic states by evictinghistones from open chromatin, which occurs irrespective of its abilityto induce DNA breakage, and was shown to alter the transcriptome ofcancer cells; however, the consequences of these effects are largelyunknown. Despite using chemotherapeutic drugs for more than half acentury, skilled artisans still don't fully understand their mechanismof action or why they preferentially kill cancer cells. As this currentstudy demonstrates, understanding these effects and using drugs based onthis understanding will allow for a more rational treatment of cancer.Regarding DXR, despite its success, its use is limited due to severecardiotoxicity, necessitating a maximum life-time dosage, and othercytotoxic effects. Relapse is common in T-ALL, which carries a poorprognosis and is not improved by intensified chemotherapy. PI3Kactivating mutations are common in ALL, and relapsed pediatric patientsoften show additional activation of the Wnt pathway, frequentlyresulting from epigenetic changes. Thus, repurposing DXR as a targetedtherapy against chemoresistant cells could avoid severe toxicity andreduce relapse. DXR nanoparticles are particularly well-suited for thiseffect due to their slow, sustained release and tissue distribution,which has been shown to be preferential to tumors but is markedlyreduced in the heart and other vital organs (Tran, T. H. et al. Longcirculating self-assembled nanoparticles from cholesterol-containingbrush-like block copolymers for improved drug delivery to tumors.Biomacromolecules 15, 4363-4375 (2014), which is incorporated byreference herein).

Considering the critical role of Wnt/β-catenin and PI3K/Akt cooperativesignaling in normal HSC self-renewal, inhibition pS⁵⁵²-β-catenin wasexpected to have a detrimental effect on normal HSCs, and was expectedto need to rescue treated mice with HSC transplantation, perhaps usingestablished ex vivo HSC expansion system. Unexpectedly, [Low]DXRtreatment allowed for and even facilitated recovery of HSPCs. Whiteoutbeing bound to a particular theory, it's possible that, similar to thephenomenon of oncogene addiction, LSCs are ‘addicted’ to the β-cat/Aktmechanism of self-renewal, while HSPCs may be more flexible in usingalternative pathways. Elimination of LSCs and blast cells reducescompetition, allowing for subsequent recovery of HSPCs. As LSCs competewith HSPCs for niche occupancy, elimination of LSCs in particularfacilitates a net recovery of HSPCs. The disclosed model system is idealfor future studies regarding this dynamic competition between LSCs andHSPCs, particularly regarding normal and tumorigenic niches in responseto different treatments.

Similar gene expression signatures found in cancer stem cells and normalstem cells are predictive of clinical outcomes. Clinical evidence alsoshows that rare cells with self-renewal capacity often survivechemotherapeutic treatment, indicating that self-renewal may be acommon, central property that, if targeted, would lead to more durablecures. The disclosure supports this potential and demonstrates a dynamicrelationship between chemoresistant LSCs, bulk leukemic cells, andHSPCs. Targeting tumorigenic cells discretely from their bulk progenyand preferentially over normal stem/progenitor cells would substantiallyimprove patient outcomes if translated to the clinic.

Example 6

Leukemic mice for the studies described here were obtained as follows.Whole bone marrow was isolated from uninduced Scl-Cre positivePten:β-catAct mice and mixed with an equal portion of congenic,wild-type bone marrow, and transplanted into irradiated (10 Gy) Ptprcrecipient mice. 4-6 weeks post-transplant, recipient mice were placed ontamoxifen feed for 2 weeks in order to induce recombination, whichresulted in leukemia development by 7 weeks post-induction in allrecipient mice. 7 weeks post-induction, leukemic mice were injectedintravenously (via the tail vein) with 5 doses (once per dayconsecutively) of 0.5 mg/kg Doxorubicin (‘[Low]Doxo’) or given a singleinjection (day 1 only) of doxorubicin nanoparticles (single 0.8 mg/kginjection) (‘[Low]NanoDoxo’) or a single injection (day 1 only) ofDoxil® (single 0.8 mg/kg injection). Chemotherapy was used concurrently(once per day for 5 consecutive days) and consisted of Nelarabineinjected intravenously at 217 mg/kg and Dexamethasone (intraperitonealinjection) at 10 mg/kg. All drugs were solubilized in 45%(2-Hydroxypropyl)-β-cyclodextrin (HBC). Doxorubicin hydrochloride(Sigma; D1515) with or without Nelarabine (Selleck) was dissolved in HBC(43.4 mg/ml Nelarabine +/−0.1 mg/ml Doxorubicin) and administered IVaccording to the formula: Body Weight (g)×5=volume to inject (μl).Dexamethasone (BioVision) was dissolved in HBC at 2.5 mg/ml and injectedIP according to the formula: Body Weight (g) ×4=volume to inject (μl).

The doxorubicin nanoparticles have an average particle diameter of 138nm. The drug loading reached as high as 22.1% (w/w). The release of Doxin PBS was steady at approximately 2% per day, with 24% released in 12days. The nanoparticles significantly increased the circulation time ofthe drug in blood compared to free Dox. Pharmacokinetics andbiodistribution of nanoparticle-based Dox in mice bearing subcutaneoustumors showed higher blood concentrations and lower accumulation inheart, lung, kidney, spleen and liver compared with free Dox at 24 hafter intravenous injection, indicating a much greater safety profile(FIG. 15a-b ). Cardiotoxicity was tested based on cardiac troponin Ilevel and histology. No heart damage was identified after treating withdoxorubicin nanoparticles (5 mg/kg once per week) for 2 months, whilefree Dox at 1 mg/kg (8 doses) and high single dose showed cardiotoxicity(FIG. 15-c).

FIG. 14 shows that doxorubicin nanoparticles have enhanced effectivenessin eliminating leukemic stem cells and facilitating normal hematopoieticstem/progenitor cell recovery compared to Doxil®. While cytotoxicchemotherapy induces LSC expansion, low-dose doxorubicin administereddaily for five days prevents this expansion and even facilitatesrecovery of HSPCs. Doxorubicin nanoparticles allow for further reduceLSCs compared to free doxorubicin and effectively eliminate thispopulation. This effect is obtained through only a single low-doseinjection on day 1. Doxil® is not as effective at preventing the LSCexpansion induced by chemotherapy or at facilitating HSPC recovery asDoxorubicin Nanoparticles.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be incorporated within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated herein by referencefor all purposes.

1. A method of treating cancer, comprising administering to a subject inneed thereof a pharmaceutically active molecule that is capable ofselectively inhibiting p-S⁵⁵²-β-catenin, p-T²¹⁷-β-catenin,p-T³³²-β-catenin, and/or p-S⁶⁷⁵-β-catenin production and/or activity,wherein the pharmaceutically active molecule is administered in anamount effective to reduce and/or limit cancer-initiating cells.
 2. Themethod of claim 1 wherein the cancer is resistant to traditionaltreatment.
 3. The method of claim 2, wherein the traditional treatmentis radiation therapy, chemotherapy, immunotherapy, or any combinationthereof.
 4. The method of claim 1, wherein the cancer is selected fromthe group consisting of leukemia, lymphoma, prostate cancer, breastcancer, endometrial cancer, gastrointestinal cancer, lung cancer,melanoma, sarcoma, neuroblastoma, mesothelioma, testicular cancer,thyroid cancer, ovarian cancer, uterine cancer, pancreatic cancer, livercancer, and Wilms' Tumor.
 5. (canceled)
 6. The method of claim 1,wherein the pharmaceutically active molecule is administered in a lowdose.
 7. The method of claim 6, wherein the low dose is about ⅕ to 1/50of the clinical dose of the pharmaceutically active molecule when dosedfor chemotherapy.
 8. The method of claim 1, wherein the pharmaceuticallyactive molecule is administered in a nanoparticle formulation. 9-13.(canceled)
 14. The method of claim 1, wherein the pharmaceuticallyactive molecule is administered in one or more nanoparticle compositionscomprising a block copolymer in a core/shell form, wherein the blockcopolymer comprises: a first block, which is of formula:

and a second block, which is of formula:

wherein m and n are independently an integer about 3 to about 500; A isindependently selected from polynorbonene, polycyclopentene,polycyclooctene, polyacrylate, polymethacrylate, a polysiloxane,polylactide, polycaprolactone, polyester, and polypeptide; R₁ is asteroid moiety optionally comprising a linker; and R2 is a polyalkyleneoxide moiety. 15-21. (canceled)
 22. The method of claim 14, comprisingthe structure:

wherein x is an integer between about 3 and about 100; m is an integerbetween about 5 and about 200; and n is an integer between about 5 andabout
 100. 23-26. (canceled)
 27. The method of claim 1, wherein thepharmaceutically active molecule is administered in one or morenanoparticle compositions comprising a block copolymer in a core/shellform, wherein the block copolymer comprises: a first block, which is offormula:

and a second block, which is of formula:

wherein q is an integer about 3 to about 500; A¹ is independentlyselected from polyacrylate, polymethacrylate, polynorbonene,polycyclopentene, polycyclooctene, polysiloxane, polylactide,polycaprolactone,polyester, and polypeptide; R¹¹ is a steroid moietyoptionally comprising a linker R¹⁴; R¹² is polyalkylene oxide,polyester, or polypeptide moiety; and R¹³ is a disulfide linker moiety.28-32. (canceled)
 33. The method of claim 27, wherein the first block isof formula:

34-36. (canceled)
 37. The method of claim 27, wherein R¹³ is of formula:

38-40. (canceled)
 41. The method of claim 27, comprising the structure:

wherein wherein X is a trithiocarbonate, dithiocarbamate, ordithioester; q is an integer between about 5 and about 200; and r is aninteger between about 5 and about
 100. 42-44. (canceled)
 45. The methodof claim 8, wherein the pharmaceutically active molecule is hydrophobic.46. The method of claim 8, wherein the pharmaceutically active moleculeis anthracycline.
 47. The method of claim 8, wherein thepharmaceutically active molecule is doxorubicin, daunorubicin,vincristine, epirubicin, idarubicin, valrubicin, mitoxantrone,paclitaxel, docetaxel, cisplatin, camptothecin, irinotecan,5-fluorouracil, methotrexate, or dexamethasone.
 48. (canceled) 49.(canceled)
 50. The method of claim 8, comprising administering acombination of two different nanoparticle compositions.
 51. (canceled)52. The method of claim 50, wherein a second nanoparticle compositioncomprises the hydrophobic pharmaceutically active molecule selected fromthe group consisting of daunorubicin, vincristine, epirubicin,idarubicin, valrubicin, mitoxantrone, paclitaxel, docetaxel, cisplatin,camptothecin, irinotecan, 5-fluorouracil, methotrexate, anddexamethasone.
 53. (canceled)
 54. The method of claim 8, wherein thenanoparticle composition further comprises one or more metalnanoparticles or quantum dots.
 55. (canceled)
 56. (canceled)
 57. Themethod of claim 8, wherein the nanoparticle is between about 5 and about500 nm in diameter.
 58. (canceled)
 59. (canceled)